Based on the limited heterozygosity and strong linkage disequilibrium reported for several Leishmania species, it has been proposed that these parasites are essentially clonal. This notion must be reconciled, however, with the accumulating examples of naturally occurring strains that share genotypic markers from two recognized species, providing circumstantial evidence for sexual recombination. We have been able to provide conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle closely resembling the meiotic process of African trypanosomes. Two clonal L. major parental lines were studied, each bearing a different drug resistance gene integrated into distinct sites on different chromosomes. The parental clones were tested for their ability to generate parasites resistant to both drugs during co-infection in the sand fly. Of 102 flies from 4 independent co-infection experiments, 26% grew out promastigotes that were resistant to both drugs. Extensive analyses of SNPs present on chromosomes not linked to the drug resistance markers revealed that while each parent was homozygous for every marker tested, all 18 progeny tested clearly inherited both parental alleles. This provided strong evidence that each progeny clone inherited a full set of chromosomes from each parent and were thus full genome hybrids. Analysis of total DNA revealed that the parents and most hybrids showed 2n DNA contents, suggesting a Mendelian model of meiosis of the parental strains followed by fusion of the haploid cells. The few triploid offspring observed might be attributed to incomplete meiotic division of one parent and fusion of haploid and diploid cells. In contrast to the chromosomal DNA, maxicircle markers demonstrated clear and consistent uniparental inheritance. Our data suggest that sexual recombination does occur in Leishmania and likely contributes to the inter- and intra-species diversity seen in nature. Leishmania mutants deficient in genes controlling the biosynthesis of LPG and related cell surface and released glycoconjugates have provided some of the more striking phenotypes in Leishmania biology. Based largely on the analysis of these mutants, we have previously shown that a family of cell surface and secreted phosphoglycan-containing molecules are essential for Leishmania growth and development in the sand fly vector. L. major LPG2-/- mutants, which are deficient in expression of all phosphoglycan-containing molecules, showed significantly reduced survival and growth in the midguts of P. duboscqi and L. longipalpis vectors 36-72 hours after the infective feed, whereas wild type and LPG2 add back parasites developed normally. Inhibitors of serine and cysteine proteases promoted the early survival and growth of LPG2-/- in the blood meal, indicating that in the absence of phosphoglycans, the digestive enzymes induced by blood feeding are lethal to developing promastigotes. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of LPG2-/- promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function in a manner similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage. The ability of neutrophils to rapidly respond to infection and efficiently phagocytose a variety of pathogens, including L.major, suggests that these cells may also be an initial cellular target of Leishmania infection. The role of neutrophils has ever been addressed in sand fly-transmitted Leishmania infections. Using a combination of both dynamic intravital multiphoton microscopy (2P-IVM), and flow cytometric analysis, we studied the early events in the skin following sand fly or needle inoculation of L. major. Flow analysis of the cellular infiltrate into the skin initiated by sand fly feeding revealed a dramatic and sustained increase in neutrophil numbers accompanied by a significant increase in macrophages, regardless of the infectious status of the flies. A system was established for visualizing the bite site in vivo, employing an RFP-expressing strain of L.major and gene-targeted mice expressing eGFP under the control of the endogenous lysozyme M promoter. GFPhi neutrophils clearly delineated the location of proboscis penetration through both epidermal and dermal layers of the skin, and using 2P-IVM, neutrophils rapidly accumulated in, and subsequently swarmed around, the vicinity of sand fly bites at 30-60 min. after exposure to the bite. Parasite phagocytosis by neutrophils was readily observed at early time points following transmission. Following intradermal inoculation of RFP parasites by needle, 2P-IVM revealed the rapid accumulation of neutrophils inside blood vessels surrounding the infection site as early as 30 p.i. and the subsequent extravasation of these cells into the skin parenchyma. Phagocytosis occurred concurrently with neutrophil migrational arrest. Importantly, L. major captured by neutrophils at the inoculation site were viable and could transfer infection as efficiently as infectious stage parasites. Thus, our observations indicate that neutrophils are the initial host cell for a substantial fraction of parasites following infection, and the early influx and persistence of neutrophils, rather than contributing to host resistance, appears critical to the infectious process.